CN104155445B - Preparation and application of unmarked electrochemiluminescent tumor marker immunosensor - Google Patents
Preparation and application of unmarked electrochemiluminescent tumor marker immunosensor Download PDFInfo
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Abstract
The invention discloses a preparation method of an unmarked electrochemiluminescent tumor marker immunosensor and application of the immunosensor in detection of tumor markers, and belongs to the field of novel nano functional materials and biosensors. According to the preparation method of the unmarked electrochemiluminescent tumor marker immunosensor, a novel micro-nano material NH4CoPO4 is taken as a substrate so that the luminescence stability of a luminol-hydrogen peroxide luminescent system can be greatly enhanced, and meanwhile, rod-like gold@siver core-shell nanoparticles are taken as a biomimetic catalytic luminescent system, and therefore, an unmarked electrochemiluminescent immunosensor, which is low in cost, high in sensitivity, good in specificity, fast in detection and simple to prepare and is used for detecting tumor makers, can be prepared.
Description
Technical field
The present invention relates to a kind of Synthesis and applications of unmarked electrochemiluminescence tumor markers immunosensor.Specifically adopt unmarked electrochemical luminescence method, based on cobaltous ammonium phosphate flaky material (NH
4coPO
4) and the immunosensor of quick, Sensitive Detection tumor markers that builds of gold silver core-shell nanometer rod – Shandong rice promise – Chitosan Composites (Au AgNRs – Lum – Chi), belong to Nano-function thin films and biosensor technology field.
Background technology
The early diagnosis of tumour, early treatment are the keys improving cancer patient's survival rate.The detection of tumor markers can reflect the biological characteristics of tumour, all significant to treatment, auxiliary diagnosis, judging prognosis.The clinical testing procedure of current existing tumor markers is a lot, and as enzyme immunoassay, radioimmunology etc., but these detection methods exist many deficiencies, and in inactivation as easy in enzyme in enzyme immunoassay, radioimmunology, radioactive contamination is serious etc.Therefore, invention one is highly sensitive, the less demanding environmentally friendly tumor marker sensor of high specificity, condition of work is extremely urgent.
Electrochemiluminescence immunosensor is widely used in the field such as clinical diagnosis, Pharmaceutical Analysis, as tumor marker sensor etc. due to advantages such as it are highly sensitive, specificity is good, easy and simple to handle.The electrochemiluminescence immunosensor that processability is superior, its most critical technology is exactly the raising of the performances such as the effectively fixing and reappearance of luminous intensity and stability and immune molecule.
Lu meter Nuo – hydrogen peroxide (Lum-H
2o
2) luminescence system due to low cost, luminous intensity is high, to be widely applied in the analytical approach of electrogenerated chemiluminescence, but due to Lum-H
2o
2the luminescence of luminescence system needs by certain catalyzer, just can have light signal response that is quick, sensitive, high strength, therefore generally often adds horseradish peroxidase (HRP) to carry out catalytic reaction.And the use of HRP requires harsh to reaction conditions, be unfavorable for Lum-H
2o
2the universal use of luminescence system.Bar-shaped gold silver core-shell nanometer rod (Au AgNRs) is owing to having catalysis Lum-H
2o
2the effect of luminescence system, therefore can be used to replace HRP as analogue enztme, is simultaneously inorganic nano material due to it, has more wide in range service condition compared to HRP.In addition, Au@AgNRs have specific surface area large, be easy to features such as combining with various biomolecules (nucleic acid, protein and biomolecule etc.), harmless, can be applied in the preparation of electrochemiluminescimmunosensor immunosensor.
NH
4coPO
4be a kind of micro Nano material of sheet, be applied in the research of super sensor, due to its schistose texture, there is good charge-discharge characteristic, be applied in the present invention, stable Lum-H can be reached
2o
2luminescence system, and the effect increasing electrode specific surface area.
The present invention successively uses NH
4coPO
4with Au@AgNRs-Lum-Chi, electrode is modified, then utilize tumor markers antibody and with in succession the adding of the antigen of its specific binding, luminous intensity is reduced, thus achieves the structure adopting unmarked electrochemical luminescence method to detect the immunosensor of tumor markers.The present invention utilizes NH
4coPO
4to Lum-H
2o
2the stabilization of luminescence system and Au@AgNRs are to lum-H
2o
2the catalytic action of luminescence system, devise a kind of simple, fast, unmarked electrochemiluminescence immunosensor that specificity is good and highly sensitive, and be applied to the detection of tumor markers.
Summary of the invention
An object of the present invention is that the instrument and equipment avoiding existing in existing detection method is complicated, operating process is loaded down with trivial details and to shortcomings such as the skill set requirements of reviewer are high, a kind of preparation method of unmarked electrochemiluminescence tumor markers immunosensor is provided, prepared sensor has feature that is highly sensitive, high specificity, and prepare simple, easy to operate, can be used for quick, the Sensitive Detection of tumor markers;
Two of object of the present invention is prepared unmarked electrochemiluminescence tumor markers immunosensor to use new function material as analogue enztme, instead of biology enzyme, avoids the impact of biology enzyme inactivation and testing environment when actual sample detects.
The technical solution used in the present invention is as follows:
1. the preparation method of a kind of unmarked electrochemiluminescence tumor markers immunosensor of the present invention, it is characterized in that, preparation process is:
(1) preparation of golden silver-colored core-shell nanometer rod – Shandong rice promise – Chitosan Composites (Au AgNRs-Lum-Chi)
Cetyl trimethyl ammonium bromide (CTAB) solution of 20mL gold nanorods colloidal sol (AuNRs) and 20mL 0.05 ~ 0.2 mol/L is mixed, stir at 30 ~ 45 DEG C, and order adds L-AA (AA) solution of 3 ~ 6 mL 0.1 mol/L, the silver nitrate (AgNO of 0.5 ~ 1 mL 0.05 mol/L
3) NaOH (NaOH) solution of solution and 3 ~ 6 mL 0.1 mol/L, stop stirring, leave standstill 1 ~ 3h, centrifuging, is re-dispersed into product in 20mL water, obtains golden silver-colored core-shell nanometer rod colloidal sol (Au AgNRs).
By the Au@AgNRs of 3 ~ 5 mL and 3 ~ 5 mL 1 × 10
-3luminol (Lum) the solution mixing of mol/L, stir 1 ~ 3h, centrifuging, is re-dispersed into product in shitosan (CHi) solution that 5mL massfraction is 0.3 ~ 0.7 %, obtains Au@AgNRs-Lum-Chi solution.
Described AuNRs is the aqueous solution of the bar-shaped golden nanometer particle of 20 ~ 40nm length.
Described Chi solution is that Chi sterling to be joined volume fraction be prepared from the acetic acid of 1%.
(2) cobaltous ammonium phosphate flaky material (NH
4coPO
4) preparation
In 40mL water, add 2.0 ~ 4.0 g ammonium salts and 0.15 ~ 0.25 g phosphate, after dissolving completely, add 0.15 ~ 0.25 g cobalt chloride, at 30 ~ 45 DEG C, stir 10 ~ 14h, centrifuging, dry at product being placed in 50 DEG C, namely obtain NH
4coPO
4.
Described ammonium salt is selected from one of following: ammonium chloride, ammonium bromide, ammonium phosphate, ammonium dihydrogen phosphate (ADP), diammonium hydrogen phosphate.
Described phosphate is selected from one of following: ammonium phosphate, ammonium dihydrogen phosphate (ADP), diammonium hydrogen phosphate.
(3) preparation method of unmarked electrochemiluminescence tumor markers immunosensor
1) pre-service is carried out to working electrode: the alundum (Al2O3) burnishing powder polishing glass-carbon electrode of diameter 4 mm being used successively 1.0,3.0 and 0.05 μm, carry out ultrasonic cleaning with ethanol and ultrapure water successively;
2) 1) in the electrode surface that obtains drip the NH of 5 ~ 10 μ L 1 ~ 5 mg/mL
4coPO
4aqueous solution, dries under room temperature naturally;
3) 2) in the electrode surface that obtains drip the Au@AgNRs-Lum-Chi solution of 5 ~ 10 μ L, preserves in 4 DEG C of refrigerators and dry, ultrapure water cleaning, dries film forming, 4 DEG C of preservations;
4) 3) in the electrode surface that obtains drip the tumor markers antibody-solutions of 5 ~ 8 μ L 10 μ g/mL, preserve in 4 DEG C of refrigerators and dry;
5) 4) in the electrode surface that obtains drip bovine serum albumin(BSA) (BSA) solution of 4 ~ 6 μ L 100 μ g/mL, preserve in 4 DEG C of refrigerators and dry, ultrapure water cleans, and dries film forming, 4 DEG C of preservations, obtained unmarked electrochemiluminescence tumor markers immunosensor.
2. unmarked electrochemiluminescence tumor markers immunosensor of the present invention, for the detection of tumor markers, step is as follows:
1) the tumor markers antigen standard solution of concentration known is joined in the PBS buffer solution of 40 ~ 60 μ L, pH=7.0 ~ 8.0, obtained antigen mixed solution, get 5 ~ 10 μ L antigen mixed solutions drip be coated onto prepared unmarked electrochemiluminescence tumor markers immunosensor working electrode on, preserve in 4 DEG C of refrigerators and dry, after the PBS buffer solution cleaning of pH=7.0 ~ 8.0, dry, 4 DEG C of preservations;
2) using saturated calomel electrode as contrast electrode, platinum electrode as to electrode, with 1) in the working electrode assembled form three-electrode system, be connected on electrogenerated chemiluminescence equipment; Successively add the PBS buffer solution of 10 ~ 25mL pH=7.0 ~ 8.0 and the hydrogen peroxide (H of 1mL 3 ~ 6 mmol/L in a cell
2o
2) solution; By cyclic voltammetry, cyclical voltage is applied to the working electrode of assembling; According to the relation between the light signal strength of the electrogenerated chemiluminescence of gained and tumor markers antigen concentration of standard solution, drawing curve;
3) testing sample solution is replaced the standard solution of tumor markers antigen, detect according to the method for drafting of the working curve of described tumor markers antigen.
3. the Synthesis and applications of unmarked electrochemiluminescence tumor markers immunosensor of the present invention, is characterized in that described tumor markers is selected from one of following: carcinomebryonic antigen (CEA), prostate specific antigen (PSA), CA-125, CA-199, CA-242, CA-724, alpha-fetoprotein (AFP), squamous cell carcinoma antigen (SCC).
useful achievement of the present invention
(1) tumor markers immunosensor preparation of the present invention is simple, easy to operate, achieves and detects quick, sensitive, the high selectivity of sample, have market development prospect.
(2) the present invention adopts NH first
4coPO
4be applied to the preparation of electrochemiluminescence immunosensor, utilize it to Lum-H
2o
2the stabilization of luminescence system and the effect to electrode specific surface area increase, significantly improve the stability of electrochemiluminescence immunosensor, have important scientific meaning and using value.
(3) the present invention adopts Au@AgNRs – Lum – Chi to be applied to the preparation of electrochemiluminescence immunosensor first, and by the synergy of nano material, enhanced sensitivity and catalytic action, significantly improve sensitivity and the accuracy of electrochemiluminescence immunosensor, achieve the markless detection to tumor markers, there is important scientific meaning and using value.
Embodiment
embodiment 1the preparation of Au@AgNRs-Lum-Chi.
The CTAB solution of 20mL AuNRs and 20mL 0.05 mol/L is mixed, stirs at 30 DEG C, and order adds the AA solution of 3 mL 0.1 mol/L, the AgNO of 0.5 mL 0.05 mol/L
3the NaOH solution of solution and 3mL 0.1 mol/L, stop stirring, leave standstill 1h, centrifuging, is re-dispersed into product in 20mL water, obtains Au@AgNRs.
By the Au@AgNRs of 3 mL and 3 mL 1 × 10
-3the Lum solution mixing of mol/L, stir 1h, centrifuging, product being re-dispersed into 5mL massfraction is in the chitosan solution of 0.3 %, obtains Au@AgNRs-Lum-Chi solution.
Described AuNRs is the aqueous solution of the bar-shaped AuNRs of 20nm length.
Described Chi solution is that Chi sterling to be joined volume fraction be prepared from the acetic acid of 1%.
embodiment 2the preparation of Au@AgNRs-Lum-Chi.
The CTAB solution of 20mL AuNRs and 20mL 0.1 mol/L is mixed, stirs at 38 DEG C, and order adds the AA solution of 5 mL 0.1 mol/L, the AgNO of 0.8 mL 0.05 mol/L
3the NaOH solution of solution and 5mL 0.1 mol/L, stop stirring, leave standstill 2h, centrifuging, is re-dispersed into product in 20mL water, obtains Au@AgNRs aqueous solution.
By the Au@AgNRs of 4 mL and 4 mL 1 × 10
-3the Lum solution mixing of mol/L, stir 2h, centrifuging, product being re-dispersed into 5mL massfraction is in the chitosan solution of 0.5 %, obtains Au@AgNRs-Lum-Chi solution.
Described AuNRs is the aqueous solution of the bar-shaped golden nanometer particle of 30nm length.
Described Chi solution is that Chi sterling to be joined volume fraction be prepared from the acetic acid of 1%.
embodiment 3the preparation of Au@AgNRs-Lum-Chi.
The CTAB solution of 20mL AuNRs and 20mL 0.2 mol/L is mixed, stirs at 45 DEG C, and order adds the AA solution of 6 mL 0.1 mol/L, the AgNO of 1 mL 0.05 mol/L
3the NaOH solution of solution and 6mL 0.1 mol/L, stop stirring, leave standstill 3h, centrifuging, is re-dispersed into product in 20mL water, obtains Au@AgNRs.
By the Au@AgNRs of 5 mL and 5 mL 1 × 10
-3the Lum solution mixing of mol/L, stir 3h, centrifuging, product being re-dispersed into 5mL massfraction is in the chitosan solution of 0.7 %, obtains Au@AgNRs-Lum-Chi solution.
Described AuNRs is the aqueous solution of the bar-shaped golden nanometer particle of 40nm length.
Described Chi solution is that Chi sterling to be joined volume fraction be prepared from the acetic acid of 1%.
embodiment 4cobaltous ammonium phosphate flaky material (NH
4coPO
4) preparation
In 40mL water, add 2.0 g ammonium chlorides and 0.15 g ammonium phosphate, after dissolving completely, add 0.15 g cobalt chloride, at 30 DEG C, stir 10h, centrifuging, dry at product being placed in 50 DEG C, namely obtain NH
4coPO
4.
embodiment 5cobaltous ammonium phosphate flaky material (NH
4coPO
4) preparation
In 40mL water, add 3.0 g ammonium phosphate and 0.2 g ammonium dihydrogen phosphate (ADP), after dissolving completely, add 0.2 g cobalt chloride, at 35 DEG C, stir 12h, centrifuging, dry at product being placed in 50 DEG C, namely obtain NH
4coPO
4.
embodiment 6cobaltous ammonium phosphate flaky material (NH
4coPO
4) preparation
In 40mL water, add 4.0 g ammonium dihydrogen phosphate (ADP)s and 0.25 g diammonium hydrogen phosphate, after dissolving completely, add 0.25 g cobalt chloride, at 45 DEG C, stir 14h, centrifuging, dry at product being placed in 50 DEG C, namely obtain NH
4coPO
4.
embodiment 7the preparation method of unmarked electrochemiluminescence tumor markers immunosensor.
(1) pre-service is carried out to working electrode: the alundum (Al2O3) burnishing powder polishing glass-carbon electrode of diameter 4 mm being used successively 1.0,3.0 and 0.05 μm, carry out ultrasonic cleaning with ethanol and ultrapure water successively;
(2) electrode surface obtained in (1) drips the NH of 5 μ L 1 mg/mL
4coPO
4solution, dries under room temperature naturally;
(3) electrode surface obtained in (2) drips the Au@AgNRs-Lum-Chi solution of 5 μ L, preserves and dry in 4 DEG C of refrigerators, and ultrapure water cleans, and dries film forming, 4 DEG C of preservations;
(4) electrode surface obtained in (3) drips the tumor markers antibody-solutions of 5 μ L 10 μ g/mL, preserves and dry in 4 DEG C of refrigerators;
(5) electrode surface obtained in (4) drips the BSA solution of 4 μ L 100 μ g/mL, preserves and dry in 4 DEG C of refrigerators, and ultrapure water cleans, and dries film forming, 4 DEG C of preservations, obtained unmarked electrochemiluminescence tumor markers immunosensor.
Described Au@AgNRs-Lum-Chi is prepared by embodiment 1.
Described NH
4coPO
4prepared by embodiment 4.
embodiment 8the preparation method of unmarked electrochemiluminescence tumor markers immunosensor.
(1) with embodiment 7;
(2) electrode surface obtained in (1) drips the NH of 8 μ L 3 mg/mL
4coPO
4solution, dries under room temperature naturally;
(3) electrode surface obtained in (2) drips the Au@AgNRs-Lum-Chi solution of 8 μ L, preserves and dry in 4 DEG C of refrigerators, and ultrapure water cleans, and dries film forming, 4 DEG C of preservations;
(4) electrode surface obtained in (3) drips the tumor markers antibody-solutions of 6 μ L 10 μ g/mL, preserves and dry in 4 DEG C of refrigerators;
(5) electrode surface obtained in (4) drips the BSA solution of 5 μ L 100 μ g/mL, preserves and dry in 4 DEG C of refrigerators, and ultrapure water cleans, and dries film forming, 4 DEG C of preservations, obtained unmarked electrochemiluminescence tumor markers immunosensor.
Described Au@AgNRs-Lum-Chi is prepared by embodiment 2.
Described NH
4coPO
4prepared by embodiment 5.
embodiment 9the preparation method of unmarked electrochemiluminescence tumor markers immunosensor.
(1) with embodiment 7;
(2) electrode surface obtained in (1) drips the NH of 10 μ L 5 mg/mL
4coPO
4solution, dries under room temperature naturally;
(3) electrode surface obtained in (2) drips the Au@AgNRs-Lum-Chi solution of 10 μ L, preserves and dry in 4 DEG C of refrigerators, and ultrapure water cleans, and dries film forming, 4 DEG C of preservations;
(4) electrode surface obtained in (3) drips the tumor markers antibody-solutions of 8 μ L 10 μ g/mL, preserves and dry in 4 DEG C of refrigerators;
(5) electrode surface obtained in (4) drips the BSA solution of 6 μ L 100 μ g/mL, preserves and dry in 4 DEG C of refrigerators, and ultrapure water cleans, and dries film forming, 4 DEG C of preservations, obtained unmarked electrochemiluminescence tumor markers immunosensor.
Described Au@AgNRs-Lum-Chi is prepared by embodiment 3.
Described NH
4coPO
4prepared by embodiment 6.
embodiment 10unmarked electrochemiluminescence tumor markers immunosensor prepared by embodiment 1 ~ 9, for the detection of tumor markers, step is as follows.
(1) the tumor markers antigen standard solution of concentration known is joined in the PBS buffer solution of 40 μ L, pH=7.0, obtained antigen mixed solution, get 5 μ L antigen mixed solutions drip be coated onto prepared unmarked electrochemiluminescence tumor markers immunosensor working electrode on, preserve in 4 DEG C of refrigerators and dry, after the PBS buffer solution cleaning of pH=7.0, dry, 4 DEG C of preservations;
(2) using saturated calomel electrode as contrast electrode, platinum electrode as to electrode, form three-electrode system with the working electrode assembled in (1), be connected on electrogenerated chemiluminescence equipment; Successively add the PBS buffer solution of 10mL pH=7.0 and the hydrogen peroxide (H of 1mL 3 mmol/L in a cell
2o
2) solution; By cyclic voltammetry, cyclical voltage is applied to the working electrode of assembling; According to the relation between the light signal strength of the electrogenerated chemiluminescence of gained and tumor markers antigen concentration of standard solution, drawing curve;
(3) testing sample solution is replaced the standard solution of tumor markers antigen, detect according to the method for drafting of the working curve of described tumor markers antigen.
embodiment 11unmarked electrochemiluminescence tumor markers immunosensor prepared by embodiment 1 ~ 9, for the detection of tumor markers, step is as follows.
(1) the tumor markers antigen standard solution of concentration known is joined in the PBS buffer solution of 50 μ L, pH=7.4, obtained antigen mixed solution, get 7 μ L antigen mixed solutions drip be coated onto prepared unmarked electrochemiluminescence tumor markers immunosensor working electrode on, preserve in 4 DEG C of refrigerators and dry, after the PBS buffer solution cleaning of pH=7.4, dry, 4 DEG C of preservations;
(2) using saturated calomel electrode as contrast electrode, platinum electrode as to electrode, form three-electrode system with the working electrode assembled in (1), be connected on electrogenerated chemiluminescence equipment; Successively add the PBS buffer solution of 20mL pH=7.4 and the hydrogen peroxide (H of 1mL 5 mmol/L in a cell
2o
2) solution; By cyclic voltammetry, cyclical voltage is applied to the working electrode of assembling; According to the relation between the light signal strength of the electrogenerated chemiluminescence of gained and tumor markers antigen concentration of standard solution, drawing curve;
(3) testing sample solution is replaced the standard solution of tumor markers antigen, detect according to the method for drafting of the working curve of described tumor markers antigen.
embodiment 12unmarked electrochemiluminescence tumor markers immunosensor prepared by embodiment 1 ~ 9, for the detection of tumor markers, step is as follows.
(1) the tumor markers antigen standard solution of concentration known is joined in the PBS buffer solution of 60 μ L, pH=8.0, obtained antigen mixed solution, get 10 μ L antigen mixed solutions drip be coated onto prepared unmarked electrochemiluminescence tumor markers immunosensor working electrode on, preserve in 4 DEG C of refrigerators and dry, after the PBS buffer solution cleaning of pH=8.0, dry, 4 DEG C of preservations;
(2) using saturated calomel electrode as contrast electrode, platinum electrode as to electrode, form three-electrode system with the working electrode assembled in (1), be connected on electrogenerated chemiluminescence equipment; Successively add the PBS buffer solution of 25mL pH=8.0 and the hydrogen peroxide (H of 1mL 6 mmol/L in a cell
2o
2) solution; By cyclic voltammetry, cyclical voltage is applied to the working electrode of assembling; According to the relation between the light signal strength of the electrogenerated chemiluminescence of gained and tumor markers antigen concentration of standard solution, drawing curve;
(3) testing sample solution is replaced the standard solution of tumor markers antigen, detect according to the method for drafting of the working curve of described tumor markers antigen.
embodiment 13stomach neoplasms tumor markers: CA-199, CA-242 or CA-724.
A preparations and applicatio for unmarked electrochemiluminescence stomach neoplasms tumor markers immunosensor, comprises the following steps:
(1) select stomach neoplasms tumor markers, build unmarked electrochemiluminescence immunosensor according to the step described in embodiment 1 ~ 9;
(2) detect according to the step described in embodiment 10 ~ 12, the detection technique index of stomach neoplasms tumor markers is in table 1.
The detection technique index of table 1 stomach neoplasms tumor markers
embodiment 14breast cancer tumour mark: CA-125 or CEA.
A preparations and applicatio for unmarked electrochemiluminescence breast cancer tumour marker immunosensor, comprises the following steps:
(1) select breast cancer tumour mark, build unmarked electrochemiluminescence immunosensor according to the step described in embodiment 1 ~ 6;
(2) detect according to the step described in embodiment 7 ~ 9, the detection technique index of breast cancer tumour mark is in table 2.
The detection technique index of table 2 breast cancer tumour mark
embodiment 15hepatic carcinoma mark: AFP.
A preparations and applicatio for unmarked electrochemiluminescence hepatic carcinoma marker immunosensor, comprises the following steps:
(1) take AFP as hepatic carcinoma mark, build unmarked electrochemiluminescence immunosensor according to the step described in embodiment 1 ~ 9;
(2) detect according to the step described in embodiment 10 ~ 12, the linear detection range of AFP is: 0.008 ~ 28 ng/mL, detects and be limited to: 1.4 pg/mL.
embodiment 16lung cancer tumor mark: SCC.
A preparations and applicatio for unmarked electrochemiluminescence lung cancer tumor marker immunosensor, comprises the following steps:
(1) take SCC as lung cancer tumor mark, build unmarked electrochemiluminescence immunosensor according to the step described in embodiment 1 ~ 9;
(2) detect according to the step described in embodiment 10 ~ 12, the linear detection range of SCC is: 0.008 ~ 27 ng/mL, detects and be limited to: 1.4 pg/mL.
embodiment 17prostate cancer mark: PSA.
A preparations and applicatio for unmarked electrochemiluminescence prostate cancer marker immunosensor, comprises the following steps:
(1) take PSA as prostate cancer mark, build unmarked electrochemiluminescence immunosensor according to the step described in embodiment 1 ~ 9;
(2) detect according to the step described in embodiment 10 ~ 12, the linear detection range of PSA is: 0.007 ~ 27 ng/mL, detects and be limited to: 1.4 pg/mL.
embodiment 18the detection of tumor markers in human serum.
Accurately pipette human serum sample, add the tumor markers antigen standard solution of certain mass concentration, not add the human serum of tumor markers antigen for blank, carry out recovery testu, detect according to the step of embodiment 10 ~ 12, the recovery of tumor markers in working sample, testing result is in table 3.
The testing result of Diagnostic Value of Several Serum Tumor Markers in table 3 human serum
Table 3 testing result is known, and the relative standard deviation (RSD) of result is less than 3.0 %, and average recovery rate is 96.0 ~ 102%, shows that the present invention can be used for the detection of Diagnostic Value of Several Serum Tumor Markers in human serum, and highly sensitive, the high specificity of method, result accurately and reliably.
Claims (2)
1. the preparation method of unmarked electrochemiluminescence tumor markers immunosensor, it is characterized in that, preparation process is:
(1) preparation of golden silver-colored core-shell nanometer rod – Shandong rice promise – Chitosan Composites Au AgNRs-Lum-CHi
The cetyl trimethyl ammonium bromide CTAB solution of 20mL gold nanorods Sol A uNRs and 20mL 0.05 ~ 0.2 mol/L is mixed, stir at 30 ~ 45 DEG C, and order adds the L-AA AA solution of 3 ~ 6 mL 0.1 mol/L, the silver nitrate AgNO of 0.5 ~ 1 mL 0.05 mol/L
3the NaOH NaOH solution of solution and 3 ~ 6 mL 0.1 mol/L, stop stirring, leave standstill 1 ~ 3h, centrifuging, is re-dispersed into product in 20mL water, obtains golden silver-colored core-shell nanometer rod Sol A u AgNRs;
By the Au@AgNRs of 3 ~ 5 mL and 3 ~ 5 mL 1 × 10
-3the luminol Lum solution mixing of mol/L, stir 1 ~ 3h, centrifuging, product being re-dispersed into 5mL massfraction is in the shitosan CHi solution of 0.3 ~ 0.7 %, obtains Au@AgNRs-Lum-CHi solution;
Described AuNRs is the aqueous solution of the bar-shaped golden nanometer particle of 20 ~ 40nm length;
Described CHi solution is that CHi sterling to be joined volume fraction be prepared from the acetic acid of 1%;
(2) cobaltous ammonium phosphate flaky material NH
4coPO
4preparation
In 40mL water, add 2.0 ~ 4.0 g ammonium salts and 0.15 ~ 0.25 g phosphate, after dissolving completely, add 0.15 ~ 0.25 g cobalt chloride, at 30 ~ 45 DEG C, stir 10 ~ 14h, centrifuging, dry at product being placed in 50 DEG C, namely obtain NH
4coPO
4;
Described ammonium salt is selected from one of following: ammonium chloride, ammonium bromide, ammonium phosphate, ammonium dihydrogen phosphate (ADP), diammonium hydrogen phosphate;
Described phosphate is selected from one of following: ammonium phosphate, ammonium dihydrogen phosphate (ADP), diammonium hydrogen phosphate;
(3) preparation method of unmarked electrochemiluminescence tumor markers immunosensor
1) pre-service is carried out to working electrode: the alundum (Al2O3) burnishing powder polishing glass-carbon electrode of diameter 4 mm being used successively 1.0,3.0 and 0.05 μm, carry out ultrasonic cleaning with ethanol and ultrapure water successively;
2) 1) in the electrode surface that obtains drip the NH of 5 ~ 10 μ L 1 ~ 5 mg/mL
4coPO
4aqueous solution, dries under room temperature naturally;
3) 2) in the electrode surface that obtains drip the Au@AgNRs-Lum-CHi solution of 5 ~ 10 μ L, preserves in 4 DEG C of refrigerators and dry, ultrapure water cleaning, dries film forming, 4 DEG C of preservations;
4) 3) in the electrode surface that obtains drip the tumor markers antibody-solutions of 5 ~ 8 μ L 10 μ g/mL, preserve in 4 DEG C of refrigerators and dry;
5) 4) in the electrode surface that obtains drip the bovine serum albumin(BSA) BSA solution of 4 ~ 6 μ L 100 μ g/mL, preserves in 4 DEG C of refrigerators and dry, ultrapure water cleans, and dries film forming, 4 DEG C of preservations, obtains unmarked electrochemiluminescence tumor markers immunosensor.
2. the preparation method of unmarked electrochemiluminescence tumor markers immunosensor as claimed in claim 1, is characterized in that described tumor markers is selected from one of following: CEA, prostate specific antigen PSA, CA-125, CA-199, CA-242, CA-724, AFP, squamous cell carcinoma antigen SCC.
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